Pharmaceutical composition for preventing or treating Avellino corneal dystrophy using antisense oligonucleotides (ASO)

Abstract

The present invention provides an antisense oligonucleotide pharmaceutical composition for preventing or treating Avellino corneal dystrophy, which induces the suppression of R124H mutant TGFBI protein expression, its use, or a method for treating the same. In particular, the present invention selectively acts on R124H TGFBI mRNA, which is a precursor to the production of R124H mutant TGFBI protein, the causative protein of Avellino corneal dystrophy, thereby minimizing the effect on normal WT TGFBI expression and inducing the inhibition of R124H TGFBI expression, thereby enabling highly effective prevention or treatment of Avellino corneal dystrophy.

Description

[Technical Field]

[0001] The present invention relates to an ASO pharmaceutical composition that inhibits the expression of the R124H mutant TGFBI, a pathogenic protein, for the purpose of preventing or treating Avellino corneal dystrophy, its use, and its treatment method. [Background technology]

[0002] The cornea is an important eye tissue for ensuring vision and maintaining visual acuity, and corneal opacity is directly linked to decreased visual acuity and inconvenience in daily life, so it is important to maintain corneal transparency. Corneal dystrophy is a condition in which opaque deposits form in the cornea due to certain factors, causing corneal opacity and decreased visual acuity, and these symptoms gradually worsen with age.

[0003] Granular corneal dystrophy is a slowly progressing corneal disorder that usually begins in infancy and gradually leads to corneal clouding, with severe visual impairment occurring after the age of 60. It is also called granular corneal degeneration, granular keratopathy, or granular corneal dystrophy. There are two types of granular corneal dystrophy. Type 1 is a very rare type of corneal disorder. It was first described by Arthur Groenouw of Germany in 1890. Type 2 of granular corneal dystrophy (Avellino dystrophy) is also a very rare type of corneal disorder. Folberg first described the disease in 1988, and the name was taken from the first four patients, who originated in the Avellino region of Italy.

[0004] Granular corneal dystrophy is known to be caused by a point mutation in transforming growth factor s-induced (TGFBI) located on chromosome 5q31. The most common mutation is the R124H mutation (R124H TGFBI), in which arginine at position 124 in the TGFBI protein is replaced with histidine, causing granular corneal dystrophy type 2, or Avellino dystrophy.

[0005] TGFBI is a 68kDa extracellular matrix protein composed of 683 amino acids that is primarily involved in cell adhesion, migration, and differentiation. In the cornea, it is primarily expressed in corneal fibroblasts (keratocytes) and corneal epithelium cells, where it interacts with the collagen components of the corneal stroma to repair corneal wounds and form the extracellular matrix. However, unlike the normal protein, TGFBI point mutant proteins exhibit abnormal three-dimensional folding, are easily aggregated, and are poorly degraded, resulting in the accumulation of opaque insoluble deposits in the corneal stroma and Bowman's membrane, leading to granular keratopathy.

[0006] Granular corneal dystrophy type 2 (GCD 2), also known as Avellino corneal dystrophy, is an autosomal dominant genetic disease in which 1 in 870 people in Korea has the R124H TGFBI mutation. Most patients have heterozygous mutations, and the incidence increases with age, with symptoms becoming more severe. Homozygous mutants are extremely rare, occurring in approximately 1 / 1,000 or 1 / 10,000 cases compared to heterozygous mutants, and the disease begins in childhood with even more severe symptoms than heterozygous mutants.

[0007] Current treatments include corneal transplants and superficial keratectomy, which removes the corneal deposits. Corneal transplants can maintain a clear cornea for a long period of time, but they are highly invasive, require a donor, and have the risk of recurrence. Keratectomy is a minimally invasive method that preserves vision until a corneal transplant is performed, but repeated procedures are required to maintain clarity, which can lead to presbyopia due to a decrease in refractive index, and there is also a high risk of recurrence and worsening due to genetic predisposition.

[0008] Although various research efforts have been conducted on therapeutic drugs, no successful results have been achieved to date. For example, KR 10-1370659 attempted to reduce deposited proteins through autophagy induced by rapamycin or melatonin, and KR 10-1394538 attempted to reduce TGF-β protein expression using vitamin D3. US 2008 / 0267946 attempted to alleviate the symptoms of patients with Avellino corneal dystrophy, exacerbated by TGF-β induced by strong light such as ultraviolet light, using a TGF-s antibody.

[0009] However, these attempts to date have not led to the development of successful drugs, and the mechanism itself has been limited by its lack of direct connection to the causative agent, R124H TGFBI. In particular, if the causative agent, R124H TGFBI, aggregates in the cornea, it is difficult to dissolve or remove with drugs, and surgery can only result in new aggregation. Therefore, it is necessary to develop a successful drug for treating Avellino corneal dystrophy by developing a therapeutic agent that specifically targets and inhibits R124H TGFBI as a way to block the aggregation of the causative protein at an earlier stage.

[0010] Antisense oligonucleotides (ASOs) are single-stranded oligodeoxynucleotides that specifically bind to mRNA with a complementary sequence and can suppress the expression of target proteins by either inducing the RNA-degrading enzyme RNase H to degrade the mRNA (degradation) or blocking its translation to protein (translation blocking). The present invention aims to provide a method for fundamentally treating or preventing Avellino corneal dystrophy induced by R124H TGFBI using an ASO that specifically inhibits the expression of R124H TGFBI, the causative protein of Avellino corneal dystrophy. Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to provide a pharmaceutical composition for preventing or treating Avellino corneal dystrophy by inhibiting the expression of R124H TGFBI using ASO, its use, and its treatment method.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, which uses ASO to specifically inhibit R124H mutant TGFBI while minimizing the impact on normal TGFBI (wild-type TGFBI) expression, as well as its use and treatment method. [Means for solving the problem]

[0013] To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, comprising an antisense oligonucleotide having a base sequence that suppresses the expression of R124H mutant TGFBI.

[0014] The present invention also provides use of an antisense oligonucleotide having a base sequence that inhibits TGFBI expression in the R124H mutation as a preventive or therapeutic agent for Avellino corneal dystrophy.

[0015] The present invention also provides a method for preventing or treating Avellino corneal dystrophy, comprising administering an antisense oligonucleotide having a base sequence that suppresses the expression of R124H mutant TGFBI. [Effects of the Invention]

[0016] The development of a gene therapy agent is necessary for the effective treatment of Avellino corneal dystrophy, a genetic disease with extremely high unmet medical needs. In particular, if the R124H TGFBI protein, which causes the disease, aggregates in the cornea, it is difficult to dissolve or remove with drugs, and surgery can actually trigger new aggregation. To block the cause of the disease, the present invention provides the technical advantage of using the R124H TGFBI ASO to act at the mRNA stage, a stage prior to the production of the R124H TGFBI protein, the causative protein of Avellino corneal dystrophy, and thereby suppress protein expression, thereby preventing or treating Avellino corneal dystrophy. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows the coding mRNA sequence and the corresponding amino acid sequence of the position including amino acid 124 of the normal WT TGFBI protein and the mutant R124H TGFBI protein. [Figure 2] This is a diagram showing a test method for deriving ASOs that selectively inhibit R124H TGFBI using a reporter gene system that labels WT TGFBI protein expression levels or R124H TGFBI protein expression levels. [Figure 3A] After transfection of ASO at the indicated concentrations, the expression levels of WT TGFBI protein and R124H TGFBI protein were confirmed. [Figure 3B] After transfection of ASO at the indicated concentrations, the expression levels of WT TGFBI protein and R124H TGFBI protein were confirmed. [Figure 3C] After transfection of ASO at the indicated concentrations, the expression levels of WT TGFBI protein and R124H TGFBI protein were confirmed. [Figure 4] This shows the results of confirming the expression levels of WT TGFBI protein and R124H TGFBI protein after administering ASO without a transfection reagent (gymnotic delivery). [Figure 5] The results show that the expression levels of WT TGFBI protein and R124H TGFBI protein were confirmed after treatment with ASO at various concentrations without a transfection reagent. DETAILED DESCRIPTION OF THE INVENTION

[0018] While the present invention will be described in detail below with reference to preferred embodiments thereof, the scope of the present invention is not limited to the following embodiments, as the embodiments may be modified in various ways.

[0019] The present invention aims to prevent or treat Avellino corneal dystrophy by inhibiting the production of TGFBI protein, which is the cause of the disease, using a DNA-based antisense oligonucleotide (ASO), and more preferably aims to prevent or treat Avellino corneal dystrophy by selectively inhibiting the production of R124H mutant TGFBI protein among TGFBI proteins.

[0020] Avellino keratopathy (granular keratopathy II), which is the disease to be treated in this invention, is a dominant genetic disease caused by the R124H mutation of the TGFBI protein. The resulting mutant TGFBI protein does not fold normally and is unstable, forming granular deposits in the corneal stromal layer, gradually reducing corneal transparency and even causing vision loss.

[0021] The TGFBI protein associated with Avellino corneal dystrophy is made from mRNA with 2,052 nucleic acid units and 684 codons, and is ultimately translated into a protein consisting of 683 amino acids. Avellino corneal dystrophy occurs when a point mutation is induced in the 124th codon of this TGFBI CDS (coding sequence) from CGC to CAC (Figure 1). To date, there is no cure, and even with surgery, it is a serious genetic disease that easily recurs and leads to gradual vision loss.

[0022] Furthermore, most patients with Avellino corneal dystrophy have heterozygous mutations, in which both normal and mutant proteins are expressed depending on the allele, while patients with homozygous mutations, in which only the mutant protein is expressed, are extremely rare, occurring in approximately 1 / 1,000 or 1 / 10,000 cases. Therefore, while it is necessary to suppress TGFBI overall in order to develop a therapeutic agent, it is also necessary to have the ability to selectively suppress R124H mutant TGFBI while reducing the effect on wild-type TGFBI.

[0023] Therefore, the inventors realized that preventing the production of R124H TGFBI mutant protein could be a very useful therapeutic measure for treating Avellino corneal dystrophy, and completed the present invention by discovering a new ASO base sequence that inhibits the expression of R124H TGFBI protein.

[0024] Furthermore, since normal WT TGFBI protein plays a role in cell adhesion and differentiation to maintain tissue in the body, it is desirable to maintain the expression of normal WT TGFBI even when inhibiting the expression of mutant TGFBI.To this end, the present invention has discovered an additional ASO base sequence that selectively inhibits the expression of R124H TGFBI while minimizing the impact on the expression of WT TGFBI.

[0025] Therefore, the present invention provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, which comprises a single-stranded antisense oligonucleotide having a base sequence that suppresses the expression of TGFBI mRNA having the R124H mutation.

[0026] Avellino corneal dystrophy occurs when the mRNA codon for arginine (R) at position 124 of normal TGFBI protein, CGC, undergoes a point mutation to CAC. Therefore, the present invention may include antisense oligonucleotides having the base sequences shown in Tables 1 to 4 that directly inhibit mRNA so that protein translation by the R124H TGFBI mRNA does not occur.

[0027] Specifically, when the present invention includes an 18-mer antisense oligonucleotide, it may be one or more selected from nine base sequences (SEQ ID NOS: 1 to 9) containing modified deoxyribonucleic acid (DNA) linked by PS (phosphorothioate) bonds, as shown in Table 1, and nine base sequences (SEQ ID NOS: 10 to 18) containing unmodified DNA linked by PS bonds.

[0028] When a 19-mer antisense oligonucleotide is included, it may be one or more of the following: a 10-base sequence (SEQ ID NOs: 19 to 28) containing modified DNA linked by PS bonds as shown in Table 2; and a 10-base sequence (SEQ ID NOs: 29 to 38) containing unmodified DNA linked by PS bonds.

[0029] When a 20-mer antisense oligonucleotide is included, it may be one or more of the 11 base sequences (SEQ ID NOs: 39 to 49) containing modified DNA linked by PS bonds as shown in Table 3 and the 11 base sequences (SEQ ID NOs: 50 to 60) containing unmodified DNA linked by PS bonds.

[0030] Furthermore, the present invention also relates to one or more base sequences selected from the R124H TGFBI complementary sequence (SEQ ID NOs: 61 to 67) in which one or more mismatches are further added, as shown in Table 4.

[0031] ASO sequences are basically based on deoxyribonucleic acid (DNA), and when modifications are applied, they are represented as follows:

[0032] * =2'-O-Methoxyethyl (2'-MOE)

[0033] m=5-methyl

[0034] - =phosphorothioate linkage (PS bond)

[0035] [Table 1]

[0036] [Table 2]

[0037] [Table 3]

[0038] [Table 4]

[0039] In particular, the present invention provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, characterized by comprising one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 14, 17, 22, 23, 24, 43, 44, and 45 of the base sequences in Table 1 below, which inhibit R124H TGFBI expression.

[0040] Preferably, the present invention provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, characterized by comprising one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45 as base sequences that selectively inhibit R124H TGFBI expression while minimizing the effect on WT TGFBI expression, and more preferably, by comprising one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, and 45.

[0041] Meanwhile, the present invention provides a use as a preventive or therapeutic agent for Avellino corneal dystrophy, characterized in that it contains one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 14, 17, 22, 23, 24, 43, 44, and 45 as a base sequence that inhibits the expression of R124H TGFBI mRNA.

[0042] Preferably, the present invention provides a preventive or therapeutic agent for Avellino corneal dystrophy, characterized by comprising one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45 as base sequences that selectively inhibit R124H TGFBI expression while minimizing the effect on WT TGFBI expression, and more preferably, by comprising one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, and 45.

[0043] The present invention provides a method for preventing or treating Avellino corneal dystrophy, comprising administering an antisense oligonucleotide having a base sequence that inhibits R124H TGFBI expression, wherein the base sequence comprises one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 14, 17, 22, 23, 24, 43, 44, and 45.

[0044] Preferably, the present invention provides a method for preventing or treating Avellino corneal dystrophy, characterized by comprising one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45 as base sequences that selectively inhibit R124H TGFBI expression while minimizing the effect on WT TGFBI expression, and more preferably, by comprising one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, and 45.

[0045] Furthermore, although the present invention is structurally based on 18-20 mers, it would not depart from the scope of the present invention if the nucleotide sequences of SEQ ID NOs: 1 to 67 were used and 1 to 6 nucleic acids or nucleic acid derivatives were additionally added to the 5' and 3' ends of the sequences. It would not depart from the scope of the present invention if the nucleotide sequences of SEQ ID NOs: 1 to 67 were used and a biodegradable or non-biodegradable linker was added to the 5' or 3' end of the sequences, or if other compounds were added without linkage. It would not depart from the scope of the present invention if the nucleotide sequences of SEQ ID NOs: 1 to 67 were used and a biodegradable or non-biodegradable linker was added to the sugar structure, base structure, or binding site between the nucleic acids of these intermediate sequences, or if other compounds were added without linkage.

[0046] Additionally, the present invention provides a pharmaceutical composition for preventing or treating Avellino corneal dystrophy, characterized in that the sugar structure, base structure, or binding site between the nucleic acids of the antisense oligonucleotide is chemically modified.

[0047] That is, the antisense oligonucleotides according to the present invention may be modified in various ways as conceivable by those skilled in the art, and such modifications are within the scope of the present invention as long as the base sequence itself remains unchanged. Here, the degree to which the base sequence itself remains unchanged means that at least about 90% of the base sequence is maintained, even if a portion of the 5'-end or 3'-end of the base sequence or a portion of the intermediate sequence is added, replaced, or deleted.

[0048] As an example of modification, even if the binding site between nucleic acids has modifications such as phosphorothioate, thiophosphoramidate, phosphonate or methylphosphonate, it does not depart from the scope of the present invention as long as the base sequence is not changed. Furthermore, chemical modifications to nucleic acid structures, such as 2'-O-Methoxyethyl (2'-MOE), 2'-Fluoro, 2'-O-Methyl (2'-OMe), LNA (locked nucleic acid), PNA (peptide nucleic acid), PMO (phosphoramidate morpholino oligomer), N3'-P5' phosphoramidates, 2'-deoxy-2'-fluoro-β-D-arabino nucleic acid analogue (FANA), cyclohexene nucleic acids (CeNAs), tricyclo-DNA (tcDNA), 2-thiothymidine, 3'-fluorohexitol nucleic acid (FHNA), ethylene-bridged nucleic acid (ENA), and (S)-constrained ethyl (cEt) modifications, do not depart from the scope of the present invention, as long as the base sequence remains unchanged. Modifications such as the addition of 5-methyl pyrimidine to the base moiety, or the addition of additional fluoro groups, also do not depart from the scope of the present invention.

[0049] The pharmaceutical composition according to the present invention may be provided in one or more dosage forms selected from the group consisting of gel, emulsion, injection, and aerosol by a conventional method, but is not limited thereto.

[0050] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of carriers and diluents commonly used in the manufacture of pharmaceutical compositions, antioxidants, buffers, bacteriostatic agents, dispersing agents, and surfactants.

[0051] Specifically, the carrier and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, polyvinylpyrrolidone, or water. In addition, non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, ethyl oleate, and synthetic oils.

[0052] The pharmaceutical compositions can be administered to a subject in the usual manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, intraocular or intradermal routes.

[0053] The desired dosage of the antisense oligonucleotide varies depending on the condition and weight of the subject, the type and severity of the disease, the drug form, the route and duration of administration, and can be appropriately selected by those skilled in the art, and the scope of the present invention is not limited thereby.

[0054] In the present invention, the "subject" is a mammal, including a human, but is not limited to these examples.

[0055] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0056] Examples 1 to 67: Preparation of antisense oligonucleotides targeting R124H TGFBI mRNA

[0057] Antisense oligonucleotides for each SEQ ID NO were prepared using an oligo synthesizer. They were synthesized according to the sequence notation in Table 5, with modifications based on DNA.

[0058] <Applied transformation>

[0059] * =2'-O-Methoxyethyl (2'-MOE)

[0060] m=5-methyl

[0061] - =phosphorothioate linkage (PS bond)

[0062] [Table 5-1] [Table 5-2] [Table 5-3]

[0063] Experimental Example 1: Construction of a reporter gene system for evaluating the ability to inhibit the expression of human WT TGFBI and human R124H TGFBI

[0064] To develop an ASO that selectively acts on R124H TGFBI compared to WT TGFBI, we constructed a system in which a reporter gene was co-expressed with the protein expression of WT TGFBI or R124H TGFBI. As shown in Figure 1, the human WT TGFBI CDS or human R124H TGFBI CDS was inserted into the psiCHECK-2 vector (Promega) to allow expression of the reporter gene, Renilla luciferase, during protein expression of human WT TGFBI or human R124H TGFBI. The vector also contained a control reporter gene, Firefly luciferase, which is expressed upon transfection of the plasmid. This gene was designed to normalize the level of transfection of the plasmid and thereby enable accurate and reproducible results regarding the effect on the expression of the target gene, WT TGFBI or R124H TGFBI.

[0065] Experimental Example 2: Evaluation of inhibitory activity against human WT TGFBI and human R124H TGFBI expression (evaluation through ASO transfection)

[0066] The plasmids of Experimental Example 1 (psi-CHECK2_WT TGFBI and psi-CHECK2_R124H TGFBI) were transfected into HEK-293 cells with very low levels of endogenous TGFBI expression, and the evaluation of the examples was carried out. HEK-293 cells were transfected into a 96-well plate at 1.7 x 10 4 The cells were dispensed at 100 cells / well and cultured overnight in DMEM (Thermo Fisher Scientific) containing 10% FBS at 37°C and 5% CO. The psi-CHECK2_WT TGFBI plasmid or psi-CHECK2_R124H TGFBI plasmid was transfected into different wells of a 96-well plate at 20 ng per well using lipofectamine 2000 (Thermo Fisher Scientific). Six hours later, the ASO of the present example was transfected at 0, 25, 50, or 100 nM per well using lipofectamine RNAiMAX (Thermo Fisher Scientific). Twenty-four hours after ASO treatment, luminescence due to the expression of Renilla luciferase and Firefly luciferase was measured using a plate reader (GloMax, Promega) with the Dual-Glo luciferase assay system (Promega). The Renilla luciferase luminescence value measured for each well was divided by the Firefly luciferase luminescence value for correction. The value for the untreated control group was set to 1, and the values ​​for the ASO-treated group were expressed relative to each other, as shown in Figure 3.

[0067] As a result, the ASOs that inhibited R124H human TGFBI expression by 50% or more at a concentration of 25 nM were Examples 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45, each containing the base sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45. Furthermore, the ASOs that inhibited R124H human TGFBI expression by 50% or more at a concentration of 25 nM but were unable to inhibit WT TGFBI expression by 50% or more at the same concentration were Examples 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45, each containing the base sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45. Furthermore, the ASOs that inhibited R124H human TGFBI expression by more than 50% at a concentration of 25 nM but were unable to inhibit WT TGFBI expression by more than 50% at 50 nM were Examples 2, 3, 4, 5, 6, 7, 8, and 45, each of which contained the base sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, and 45. The ASOs that inhibited R124H human TGFBI expression by more than 50% at a concentration of 25 nM but were unable to inhibit WT TGFBI expression by more than 50% at 100 nM were Examples 2, 3, 4, 5, 6, and 7, each of which contained the base sequence of SEQ ID NOs: 2, 3, 4, 5, 6, and 7.

[0068] In addition to the above sequences, the ASOs that inhibited R124H human TGFBI expression by more than 50% at a treatment concentration of 50 nM were Examples 1 and 14, which were unable to inhibit WT TGFBI expression by more than 50% at the same concentration, and in this case, each base sequence contained the sequences of SEQ ID NOs: 1 and 14.

[0069] In addition to the above sequences, the ASO that inhibited R124H human TGFBI expression by more than 50% at a treatment concentration of 100 nM was Example 17, which was unable to inhibit WT TGFBI expression by more than 50% at the same concentration, and in this case, the base sequence included the sequence of sequence number 17.

[0070] In addition, the ASOs whose R124H human TGFBI expression inhibition rate was 20% or more higher than that of WT human TGFBI regardless of the treatment concentration were Examples 1, 2, 3, 4, 5, 6, 7, 8, 14, 22, 23, 24, 38, 43, 44, 45, 61, 63, 66, and 67, and in these cases, the base sequences included the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 14, 22, 23, 24, 38, 43, 44, 45, 61, 63, 66, and 67.

[0071] In addition, the ASOs in Examples 2, 3, 4, 5, 38, 45, 61, 66, and 67 had R124H human TGFBI expression inhibition rates that were 30% or more higher than WT human TGFBI inhibition rates regardless of the treatment concentration, and in these cases, the base sequences included the sequences of SEQ ID NOs: 2, 3, 4, 5, 38, 45, 61, 66, and 67.

[0072] On the other hand, experiments were also performed on ASOs 9, 10, 13, 16, 32, 33, 35, and 38. The results showed that ASOs 9, 10, 13, and 16, which have sequences with 18mers or less, and ASOs 32, 33, 35, and 38, which have sequences with 19mers or more, had inhibitory activity against R124H mutant TGFBI that was less than 50%. Furthermore, there was little difference in inhibitory activity against wild-type TGFBI and R124H mutant TGFBI, and in some cases, wild-type TGFBI was even further inhibited (Figure 3).

[0073] Experimental Example 3: Evaluation of inhibitory activity against human WT TGFBI and human R124H TGFBI expression (ASO Gymnotic Delivery ( Evaluation through gymnotic delivery

[0074] The degree of expression inhibition of WT TGFBI and R124H TGFBI was further evaluated through gymnotic delivery, in which ASOs reach cells by themselves without transfection reagents or other delivery aids. 1.7x10 cells were cultured in a 96-well plate of HEK-293 cells. 4The cells were then aliquoted at 100 cells / well and cultured overnight in DMEM medium containing 10% FBS at 37°C and 5% CO2. The psi-CHECK2_WT TGFBI plasmid or psi-CHECK2_R124H TGFBI plasmid was transfected into separate wells of a 96-well plate at 20 ng per well using lipofectamine 2000 (Thermo Fisher Scientific). After 6 hours, the cells were treated with the ASOs from Examples 3, 4, 5, 22, 23, 24, 43, 44, and 45 diluted in culture medium at 0 and 200 nM per well. Twenty-four hours after ASO treatment, luminescence due to the expression of Renilla luciferase and firefly luciferase was measured using a Dual-Glo luciferase assay system (Promega) with a plate reader (GloMax, Promega). The luminescence value for Renilla luciferase measured for each well was divided by the luminescence value for Firefly luciferase for correction, and the value for the control group not treated with ASO was set to 1. The values ​​for the ASO-treated group were then expressed relative to each other, as shown in Figure 4.

[0075] As a result, the ASOs that inhibited R124H human TGFBI expression by 50% or more were Examples 3, 4, 5, 22, 23, 24, 43, 44, and 45, each of which contained the base sequence of SEQ ID NOs: 3, 4, 5, 22, 23, 24, 43, 44, and 45. Furthermore, the ASOs that inhibited R124H human TGFBI expression by 50% or more but were unable to inhibit WT TGFBI expression by 50% or more were Examples 3, 4, 5, 22, 23, and 45, each of which contained the base sequence of SEQ ID NOs: 3, 4, 5, 22, 23, and 45.

[0076] In addition, the ASOs in Examples 3, 4, 5, 22, 23, 24, 44, and 45 had expression inhibition rates of R124H human TGFBI that were 20% or more higher than those of WT human TGFBI, and in these cases, the base sequences included the sequences of SEQ ID NOs: 3, 4, 5, 22, 23, 24, 44, and 45.

[0077] In addition, the ASOs in Examples 3, 4, 5, 22, 23, and 45 had expression inhibition rates of R124H human TGFBI that were 30% or more higher than that of WT human TGFBI, and in these cases, the base sequences included the sequences of SEQ ID NOs: 3, 4, 5, 22, 23, and 45.

[0078] Additionally, in the same experimental procedure, Examples 5 and 45 were treated at various concentrations from 0.78 nM to 100 nM, and the IC50 for 50% inhibition of expression of WT TGFBI or R124H TGFBI was calculated. 50 was derived using GraphPad Prism 9.5.1.

[0079] As a result, as shown in Figure 5, both Example 5 and Example 45 showed higher expression inhibition rates for R124H TGFBI than for WT TGFBI across the entire treatment concentration range. 50 At the time of derivation, IC for R124H TGFBI in Example 5 50 10.56 nM, IC against WT TGFBI 50 The IC for R124H TGFBI in Example 45 was determined to be 5.71 nM. 50 IC50 of 5.71 nM, relative to WT TGFBI 50 The selectivity for R124H TGFBI over WT TGFBI was determined by the IC50 of 109.3 nM. 50 IC against R124H TGFBI 50 When expressed as a value obtained by dividing the WT / Mut value (WT / Mut ratio), Example 5 showed excellent selectivity of about 91.7 times, and Example 45 showed excellent selectivity of about 19.1 times.

[0080] [Table 6]

[0081] Comprehensive analysis of the above test results revealed that one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 14, 17, 22, 23, 24, 43, 44, and 45 are useful for inhibiting R124H TGFBI expression as ASOs that inhibit R124H human TGFBI expression by 50% or more at the total concentrations tested in Experimental Example 2.

[0082] Preferably, in Experimental Example 2, it was analyzed that one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45 are useful ASOs that inhibit R124H human TGFBI expression by more than 50% at the lowest concentration of 25 nM, but are unable to inhibit WT TGFBI expression by more than 50% at the same concentration, minimizing the effect on WT TGFBI expression and selectively inhibiting R124H TGFBI expression.

[0083] More preferably, the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, and 45 is a union of ASOs that inhibit R124H human TGFBI expression by more than 50% at the lowest concentration of 25 nM in Experimental Example 2 but are unable to inhibit WT TGFBI expression by more than 50% at the step concentration of 50 nM, and ASOs that inhibit R124H human TGFBI expression by more than 50% through gymnotic delivery (200 nM) in Experimental Example 3 but are unable to inhibit WT TGFBI expression by more than 50%. It was analyzed that one or more antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, and 45 are useful for selectively inhibiting R124H TGFBI expression while minimizing the effect on WT TGFBI expression.

[0084] Although the specific details of the present invention have been described above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention, and the substantial scope of the present invention is defined by the claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating Avellino corneal dystrophy, comprising one or more oligonucleotides selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 14, 17, 22, 23, 24, 43, 44, and 45, which are single-stranded antisense oligonucleotides having a base sequence that suppresses the expression of TGFBI having the R124H mutation.

2. The pharmaceutical composition for preventing or treating Avellino corneal dystrophy according to claim 1, characterized in that the antisense oligonucleotide comprises one or more oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, 24, 43, 44, and 45.

3. The pharmaceutical composition for preventing or treating Avellino corneal dystrophy according to claim 1, characterized in that the antisense oligonucleotide comprises one or more oligonucleotides selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 22, 23, and 45.

4. The pharmaceutical composition for preventing or treating Avellino corneal dystrophy according to claim 1, characterized in that the antisense oligonucleotide has a sugar structure, a base structure, or a binding site between one or more ribonucleic acids chemically modified.

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